Therapeutic and diagnostic miRNAs in cardioprotection

Ischemic heart disease is a leading cause of morbidity and mortality in the EU, including Austria and Hungary. Therefore, development of cardioprotective strategies to limit the size of infarction during ischemia/reperfusion injury is of great importance. Ischemic postconditioning (IPoC), a cardioprotective intervention performed as intermittent periods of coronary artery occlusions at the onset of reperfusion, has been successfully applied in humans. Little is known on the cellular mechanisms of IPoC. The role of the novel family of regulatory RNAs, the microRNAs (miRNAs) has not been investigated in IPoC. Therefore, the key goals of the present project are (1) to identify miRNAs that play a role in the mechanism of IPoC-induced cardioprotection in a clinically relevant myocardial pig infarction model by miRNA microarray and network analysis. Although IPoC can protect remote organs as well, the mechanism of this remote action is not known. MiRNAs are enriched in 50-100 nm vesicles released by most cells, named exosomes. Therefore, we aim to study (2) whether cardioprotective miRNAs are transferred by exosomes. After identification of potential cardioprotective miRNAs we aim (3) to validate the concept by assessment whether intracoronary administration of selected set of miRNAs identified as cardioprotective, reduces infarct size. In addition, (4) we also plan to identify miRNAs that can be utilized as diagnostic biomarkers for the efficacy of the cardioprotection by IPoC. The expected results include the proof of concept and mechanism of the miRNA-based cardioprotective therapy in a clinically relevant large animal model that can be translated directly to human studies.

Short repeated periods of occlusion/reperfusion after the reopening of the infarct-related artery represent ischemic postconditioning (IPostC), inducing cardioprotection thereby reducing infarct size. The mechanisms behind the cardioprotective effect of IPostC still little debated. Here, we have investigated the 1) effect of IPostC on the changes of miRNA and mRNA expression in ischemic and non-ischemic (remote) myocardium in porcine closed-chest reperfused acute myocardial infarction (AMI) model utilizing miRNA array and next generation sequencing (NGS), and 2) identified sets of miRNAs and mRNAs playing role in cardioprotection. This animal model used in these experiments is the closest to the human acute myocardial infarction with reperfusion, achieved by percutaneous coronary intervention. Domestic pigs (n=56) underwent induction of AMI by 90-min coronary balloon occlusion of the mid LAD followed by deflation of the balloon (reperfusion). The pigs were randomized to ischemic non-conditioning and IPostC groups. IPostC was performed immediately after initiation of reperfusion by repeated 6x30sec coronary balloon inflation/deflation. Sham- operated pigs served as control. Gene expression analysis of the heart was performed in remote and infarcted area by using high throughput PCR and network dynamical stimulation and attractor landscape analyses and next generation sequencing (NGS), after 3 hours or 3 days post-AMI. We assessed LV function at baseline, three hours and three-day follow-up by echocardiography and at three days follow-up by cardiac magnetic resonance imaging (cMRI). In addition, we evaluated the release of chemotactic cytokines. Compared to MI, IPostC was associated with protection on a microvascular level with attenuated oedema and microvascular obstruction. However, opposite to the expectation, IPostC did not influence the infarct size and left ventricular performance. IPostC induced several de-regulation of miRNA expression, with identification of several miRNAs mediating cardioprotection. The expression of genes involved in pro-survival kinase pathway were mostly similar in IPostC and MI experimental groups in both time windows and tissue regions. However, we observed significant deregulation of gene sets enriched in focal adhesion pathway in the late window of cardioprotection after IPostC. Transcripts involved in adhesion and activation of blood cells, cardiac hypertrophy and cardiomyopathy were downregulated, indicating a beneficial effect of IPostC on microvasculature. However, these gene expression changes did not reach the level of effective translation into clinical benefit. In conclusion, the potential cardioprotective effect of IPostC is attributed to changes in specific sets of miRNA and mRNA expression. Exploiting the complexity of the genetic response to ischemic/reperfusion injury may help to identify biomarkers or further therapeutic agents against ischemic burden of the heart.